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Epitope mapping of antibodies directed against the human neutrophil alloantigen 3a
Author(s) -
Berthold Tom,
Wesche Jan,
Kuhnert Kathleen,
Fürll Birgitt,
Hippe Holger,
Hoppen Jens,
Reil Angelika,
Muschter Stefan,
Bux Jürgen,
Greinacher Andreas
Publication year - 2011
Publication title -
transfusion
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.045
H-Index - 132
eISSN - 1537-2995
pISSN - 0041-1132
DOI - 10.1111/j.1537-2995.2011.03115.x
Subject(s) - antibody , epitope , western blot , immunoassay , peptide , immunofluorescence , epitope mapping , microbiology and biotechnology , chemistry , granulocyte , extracellular , blot , biochemistry , biology , immunology , gene
BACKGROUND: Severe transfusion‐related acute lung injury is often caused by antibodies directed against the human neutrophil alloantigen (HNA)‐3a. HNA‐3a results from an amino acid exchange (Arg154Gln) in the first extracellular loop of the choline transporter‐like protein 2 (CTL2). The characteristics of the binding domain(s) of HNA‐3a antibodies are unknown. STUDY DESIGN AND METHODS: For epitope mapping, a library of 23 different HNA‐3a (R 154 ) and three HNA‐3b (Q 154 ) peptides covering different parts of the first extracellular loop of CTL2 (aa 55‐231 ) was synthesized in Escherichia coli and tested by Western blot with two HNA‐3a alloantibody–containing plasma samples and by enzyme immunoassay (EIA) with different HNA‐3a‐ (n = 21) and HNA‐3b‐ (n = 1) positive plasma samples. RESULTS: Despite promising Western blot results using highly reactive plasma samples, we found widely varying reactivities of different HNA‐3a plasmas in the EIA, with only 11 of 21 HNA‐3a antibodies binding to any of the tested HNA‐3a peptides and with no peptide recognized by more than nine of the 21 antibodies. The HNA‐3b plasma did not react with R 154 peptides in the EIA nor with R 154 or Q 154 peptides in Western blot experiments. Plasma reactivity profiles with the peptides did not correlate with those observed using granulocyte agglutination and granulocyte immunofluorescence tests. CONCLUSION: Binding of HNA‐3a alloantibodies depends on the conformation of the intact CTL2 protein and their binding sites may differ substantially. Peptide‐based assays for detection of HNA‐3a antibodies bear the risk to be insensitive and require systematic validation with a large panel of antibodies.